Preinvasive nonhost resistance of Arabidopsis against melanized appressorium-mediated entry of multiple nonadapted Colletotrichum fungi

ABSTRACT Nonhost plants effectively block a vast number of nonadapted fungal pathogens at the preinvasive stage. On the host plants, adapted fungal pathogens such as Colletotrichum species invade into plant epidermal cell by penetration peg developed from melanized appressorium, followed by invasive hyphal extension. I reported nonadapted Colletotrichum fungi that showed an increased rate of melanized appressorium-mediated entry (MAE) into the pen2 mutant of nonhost Arabidopsis thaliana (hereafter Arabidopsis). It was also found that other MAE-type nonadapted Colletotrichum fungi with no penetration into the pen2 mutant invaded Arabidopsis in the presence of additional mutations such as edr1, gsh1, eds5, cas, and chup1 in the pen2 background. Thus, many immune components contribute to the preinvasive nonhost resistance (NHR) of Arabidopsis against Colletotrichum MAE, and PEN2-related defense takes priority over other defense pathways. Here, I show that among the above nonadapted fungi, Colletotrichum nymphaeae PL1-1-b exhibited relatively lower incompatibility with the nonhost Arabidopsis with increased MAE in each single mutant of edr1, gsh1, eds5, and cas, although other nonadapted fungi almost never invaded these single mutants. Based on the relationships between Colletotrichum MAE and the Arabidopsis immune-related components, Colletotrichum-Arabidopsis incompatibility and multilayered immunity in the preinvasive NHR of Arabidopsis are discussed in this study.

Fungal pathogens directly invade the plant epidermis, followed by the development of invasive hyphae inside; therefore, penetration into epidermal cells is the most crucial step for successful infection. Only a few adapted fungal pathogens can infect a specific plant (defined as the host). If the plant is a nonhost, a robust and broad-spectrum defense termed nonhost resistance (NHR) effectively prevents the invasion of a vast number of nonadapted fungi in incompatible interactions. 1,2 Incompatible interactions between the model brassicaceous plant Arabidopsis thaliana (hereafter Arabidopsis) and nonadapted powdery mildew fungi, such as Blumeria graminis f. sp. hordei, have been well studied for NHR. Publications reported that pen1, pen2, pen3, pmr4, ataf1, pldδ, cam7, cyp81F2, and vps9a mutant plants allowed increased entry of these obligate biotrophs into the epidermis. 3-11 PENETRATION 1 (PEN1) syntaxin mediates a secretory pathway to the fungal penetration site. 3,12 PENETRATION 2 (PEN2) myrosinase, CYTOCHROME P450 FAMILY 81 SUBFAMILY F POLYPEPTIDE 2 (CYP81F2) monooxygenase, and PENETRATION 3 (PEN3) transporter are involved in metabolism and transport of tryptophan-derived secondary metabolites, 5,6,8,13,14 while CALMODULIN 7 (CaM7) is a Ca 2+ sensor which regulates PEN3 function. 10 POWDERY MILDEW RESISTANT 4 (PMR4) callose synthase functions in wound and papillary callose formation. 4 ARABIDOPSIS THALIANA ACTIVATING FACTOR 1 (ATAF1) transcription factor suppresses abscisic acid biosynthesis and signaling. 7 PHOSPHOLIPASE Dδ (PLDδ) catalyzes phosphatidic acid production on the membrane in stress responses. 9 VACUOLAR PROTEIN SORTING 9a (VPS9a) guanine-nucleotide exchange factor is required for the delivery of membrane material to the haustorial encasement. 11 Thus, many pathways and components including as-yet-unknown factors elaborately underpin the Arabidopsis NHR against powdery mildew fungi.
The hemibiotroph Colletotrichum fungus was also instrumental in the research on NHR in Arabidopsis. Of those mentioned above, pen1, pen2, pen3, pmr4, and cyp81F2 mutants were used for the fungal entry test with nonadapted Colletotrichum tropicale S9275, a mulberry isolate, and the contribution of PEN2, PEN3, and CYP81F2 to preinvasive defense were demonstrated. 15,16 Hiruma et al. also performed screening of Arabidopsis mutants, and ENHANCED DISEASE RESISTANCE 1 (EDR1) and γ-GLUTAMYLCYSTEINE SYNTHETASE 1 (GSH1) were found as NHR contributors against entry of C. tropicale S9275 to the epidermis. 16,17 EDR1 protein kinase mediates the induction of antifungal plant defensins. 16,18 GSH1 is involved in the generation of PEN2-related metabolites through glutathione biosynthesis, while GSH1 is also required for postinvasive defense. 13,19 Immune kinases BRASSINOSTEROID INSENSITIVE 1-ASSOCIATED RECEPTOR KINASE 1 (BAK1), BOTRYTIS-INDUCED KINASE 1 (BIK1), and AVRPPHB SUSCEPTIBLE1 (PBS1)-LIKE 1 (PBL1) working in the defense signaling triggered by pathogenassociated molecular patterns were also involved in preinvasive NHR against C. tropicale S9275. 20 On all these mutant plants, however, C. tropicale S9275 could barely develop invasive hyphae through melanized appressorium, a typical infection structure of Colletotrichum fungi. Instead, this pathogen successfully entered the Arabidopsis epidermis when the carbohydrate-induced hyphal tip-based entry (HTE). [15][16][17]20 HTE, only reported in C. tropicale S9275 at present, was not associated with appressorium formation, thereby suggesting an atypical infection mode with different morphogenesis after perception of carbohydrate nutrients released from the wounded sites of the plants. 15 Therefore, there is little knowledge on Arabidopsis NHR against melanized appressoriummediated entry (MAE) into the epidermis by Colletotrichum fungi. This is in contrast to those against hemibiotroph Pyricularia oryzae (Syn. Magnaporthe oryzae), which also shows MAE during plant infection. [21][22][23][24][25][26] In P. oryzae-Arabidopsis incompatible interaction, PEN2 functions as a NHR contributor against MAE and P. oryzae slightly invaded pen2 mutant. 21 Furthermore, many other immune components such as ARABIDOPSIS and EXTRA-LARGE G PROTEIN 2 (XLG2), the single mutation of which did not affect the breakdown of penetration resistance, showed decreased preinvasive NHR in the pen2 background. [21][22][23][24][25][26] I have recently reported three nonadapted Colletotrichum fungi, C. nymphaeae PL1-1-b (MAFF240037), C. fioriniae CC1 (MAFF306550), and C. siamense MAF1 (MAFF243010), isolates from Japanese flowering cherry, cosmos, and apple, respectively, which all showed increased MAE into the pen2 mutant. 27 It was also found that two nonadapted Colletotrichum fungi, C. siamense COC4 (MAFF243696) and C. orbiculare 104-T (MAFF240422), isolates from hau tree and cucumber, respectively, could not invade pen2 single mutant, but was able to invade on pen2 mutant with additional mutations such as edr1, gsh1, eds5, cas, and chup1. 27 The MAE rates of C. fioriniae CC1 and C. siamense MAF1 into pen2 mutants were also increased in the presence of edr1, gsh1, eds5, cas, and chup1. 27 ENHANCED DISEASE SUSCEPTIBILITY 5 (EDS5) is a MATE family transporter that is required for the transport of the salicylic acid precursor outside the chloroplasts. 28,29 CALCIUM-SENSING RECEPTOR (CAS) is a Ca 2+ -sensing receptor involved in transient Ca 2+ signaling in chloroplasts during plant immunity. 30 CHLOROPLAST UNUSUAL POSITIONING 1 (CHUP1) is a regulator of epidermal chloroplast response, a newly discovered immune-related response. 27 These findings reveal that PEN2, EDR1, GSH1, EDS5, CAS, and CHUP1 all contribute to preinvasive NHR of Arabidopsis against Colletotrichum MAE. It is noteworthy that the potential effects of edr1, gsh1, eds5, cas, and chup1 mutations on the MAE of C. fioriniae CC1 and C. siamense MAF1 were actualized on the pen2 background. 27 Furthermore, quantitative real-time PCR analysis showed that many defense-related genes were induced after inoculation of C. fioriniae CC1 only in pen2 among all tested single mutants. 27 These results indicate that the PEN2-related pathway functions as a higher-layer preinvasive defense and takes priority over other defense pathways, at least in preinvasive NHR against these nonadapted MAEtype Colletotrichum fungi.
Intriguingly, among the above MAE-type nonadapted fungi (Figure 1a), C. nymphaeae PL1-1-b could partly achieve MAE in the epidermis of wild-type Arabidopsis Col-0, and the chup1 single mutant permitted an increase in the trend of the MAE rate 27 (Figure 1b and 1c). These results suggest the potential ability of C. nymphaeae PL1-1-b to overcome the higher-layer preinvasive defense(s) of Arabidopsis to a certain degree, although the PEN2-related defense pathway is still working against this pathogen 27 (Figure 1c). To clarify this point, the MAE rate of C. nymphaeae PL1-1-b into edr1, gsh1, eds5, and cas single mutants was quantified, and it was found that this pathogen showed a significant increase or increasing trend in the MAE rate in all single mutants compared to that in Col-0 ( Figure 1c). These results indicate that C. nymphaeae PL1-1-b partly overcomes the higher-layer preinvasive defense(s) in Arabidopsis and that each immune pathway related to EDR1, GSH1, EDS5, or CAS is effective against this pathogen in the wild-type plant. Thus, C. nymphaeae PL1-1-b is not adapted to Arabidopsis, but its incompatibility with the nonhost Arabidopsis is relatively low.
To visualize the relationships between Colletotrichum-Arabidopsis incompatibility and immune components in Arabidopsis preinvasive NHR, the Colletotrichum MAE rates were integrated from a previous study 27 and newly obtained data, although I did not quantified the entry rates of C. nymphaeae PL1-1-b on Arabidopsis with multiple mutations, because the effect of each mutation was already actualized in the corresponding single mutants (Figure 2). Consistent with the results of the pathogenicity assay of Colletotrichum fungi on the wild-type Arabidopsis Col-0 (Figure 1a), the MAE rates of five nonadapted fungi were notably lower than those of the adapted fungus C. higginsianum Abr1-5 (MAFF305635), a Brassicaceae pathogen (Figure 2). Five nonadapted Colletotrichum strains displayed differential ability to overcome preinvasive NHR in Arabidopsis mutants in the following order: C. nymphaeae PL1-1-b > C. fioriniae CC1 > C. siamense MAF1 > C. siamense COC4 > C. orbiculare 104-T. C. nymphaeae, C. fioriniae, and C. siamense are classified into Colletotrichum clades with broad host range (acutatum or gloeosporioides). Thus, these nonadapted strains might exhibit lower incompatibility compared to C. orbiculare 104-T which generally shows narrow host range. Since I used one representative strain from one species, except C. siamense, it is difficult to confirm that the Arabidopsis response is specific to the fungal species. On the other hand, the interaction of each nonadapted strain with the nonhost Arabidopsis provided a clear insight into preinvasive NHR in Arabidopsis. To discuss the general interactions between fungal species and Arabidopsis, it is important to evaluate the interaction of multiple fungal strains from one species with Arabidopsis.
Apart from the pen2 single mutant, the gsh1 single mutant showed a slight reduction in the preinvasive NHR against C. fioriniae CC1 and C. siamense MAF1 compared to other single mutants 27 (Figure 2). It was previously reported that GSH1 and PEN2 contribute to the same pathway in preinvasion NHR against HTE of C. tropicale S9275. 17 The slight effect of gsh1 single mutation against the MAE of these two fungi might partly reflect the link to the PEN2 pathway. GSH1 was of paramount importance for the resistance against fungal entry of C. fioriniae CC1, C. siamense MAF1, and C. siamense COC4 in the pen2 background 27 ( Figure 2); however, in the case of C. nymphaeae PL1-1-b, the gsh1 single mutant did not show a higher MAE rate than the other single mutants, suggesting that the GSH1-related pathway is not very effective against C. nymphaeae PL1-1-b (Figure 1c).
Although PEN2, EDR1, GSH1, EDS5, CAS, and CHUP1 contribute to preinvasive NHR of Arabidopsis against Colletotrichum MAE, the preinvasive NHR of the edr1 pen2 gsh1 eds5 cas chup1 hexatruple mutant against C. orbiculare 104-T still has sufficient reserves 27 (Figure 2). It is important to identify additional immune pathways that are effective against C. orbiculare 104-T and other untested nonadapted Colletotrichum strains that are highly incompatible with nonhost Arabidopsis.
PEN2 is a core NHR contributor to many fungal pathogens, including MAE-type P. oryzae and atypical HTE-type C. tropicale S9275. 5,8,13,15,21 Additionally, PEN2 conferred NHR against typical MAE-type Colletotrichum fungi 27 ( Figure 2). The use of nonadapted Colletotrichum fungi that can invade a single mutant, like pen2, through MAE, is promising; this will accelerate future research on NHR underlying Colletotrichum-Arabidopsis incompatible interactions. Currently, research on screening for NHR contributors against MAE of C. fioriniae CC1 is underway. In particular, the identification of immune component(s) other than PEN2, working in higher-layer preinvasive defenses is essential. C. nymphaeae PL1-1-b may avoid or suppress the function of such immune component(s) to partly overcome preinvasive NHR in the nonhost Arabidopsis.

Disclosure statement
No potential conflicts of interest were disclosed.

Funding
This work was supported by JSPS KAKENHI Grant Numbers 18K05643 and 21H02194, and by the Leading Initiative for Excellent Young Researchers (LEADER) program of MEXT.